REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM

描述：该项目的长期目标是了解 氮同化过程中调节基因表达的分子机制， 包括调节蛋白的作用、酶的区隔和 底物和代谢物通量在整体代谢功能中的作用 路径。脯氨酸在酿酒酵母中的利用 作为本研究的模型体系。在野外，脯氨酸是占主导地位的 这种有机体的氮源。它的使用需要耗尽 首选的但不太丰富的氮源和诱导 编码这一途径的酶的特定基因。归纳法是 由Put3蛋白介导，Put3蛋白是一种处于稳定状态的转录调节因子 在其目标基因的启动子处，保持不活跃，除非 现在时。本研究的重点是确定Put3p是如何转换的 从“关”到“开”的状态，以及它如何感觉到存在脯氨酸和 生长介质中没有首选的氮源。两位将军 将对模型进行测试：蛋白质-蛋白质或分子间相互作用 模型和构象变化或分子内相互作用模型。 Put3p是通过磷酸化和泛素化修饰的；每种修饰在 将测试该蛋白质的活性。野生型、构造型和 不可诱导的put3突变体将被检查修饰上的差异。 纯Put3p将通过质谱仪进行分析，以确定 修改。有限的蛋白分解将被用来确定Put3p 在脯氨酸存在的情况下发生构象变化。的影响 Proline对Put3p的激活是一个体外转录系统，会被 测试过。将使用双杂交技术寻找与Put3p相互作用的蛋白质 以Put3p片段为诱饵的筛选和分离 Put3p结构性突变体的抑制子。分子内相互作用将 通过使用Put3p片段的定向双杂交方法进行检测 既是诱饵又是猎物。氮素阻遏作用的五个特征 将对Pro利用系统中的调节剂进行研究。这个系统 提供了一个有用的模型来了解细胞内的变化 环境向人类新陈代谢的要素发出信号。这个 酿酒酵母在脯氨酸代谢方面的工作贡献 对最近人类编码同源基因的分离具有重要意义 酵素。这些酶的缺乏会导致高脯氨酸血症；研究 人类基因将使我们更好地理解分子基础。 这些疾病。

DESCRIPTION:The long term objective of this project is to understand molecular mechanisms that regulate gene expression in nitrogen assimilation, including the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. In the wild, proline is the predominant nitrogen source for this organism. Its utilization requires the exhaustion of preferred, but less abundant, nitrogen sources and induction of the specific genes that encode the enzymes of this pathway. Induction is mediated by the Put3 protein, a transcriptional regulator that sits poised at the promoters of its target genes, remaining inactive unless proline is present. The focus of this study is to determine how Put3p is converted from an "off" to an "on" state and how it senses that proline is present and preferred nitrogen sources are absent from the growth medium. Two general models will be tested: a protein-protein or intermolecular interaction model and a conformational change or intramolecular interaction model. Put3p is modified by phosphorylation and ubiquitination; the role of each in the activity of the protein will be tested. Wild-type, constitutive and non-inducible put3 mutants will be examined for differences in modification. Pure Put3p will be analyzed by mass spectrometry to identify sites of modification. Limited proteolysis will be employed to determine if Put3p undergoes conformational changes in the presence of proline. The effect of proline on activation of Put3p is an in vitro transcription system will be tested. Put3p-interacting proteins will be sought using two-hybrid selections employing fragments of Put3p as the bait, and by the isolation of suppressors of Put3p constitutive mutants. Intramolecular interactions will be examined by a directed two-hybrid approach using fragments of Put3p as both bait and prey. The roles of the five characterized nitrogen repression regulators in the proline utilization system will be studied. This system provides a useful model to understand how changes in the cellular environment are signaled to the elements of a metabolism in humans. The work in Saccharomyces cerevisiae on proline metabolism contributed significantly to the recent isolation of human genes encoding homologous enzymes. Deficiencies in these enzymes cause hyperprolinemias; studies on the human genes will lead to a greater understanding of the molecular basis of these diseases.